Retroviruses are extremely successful pathogens affecting virtually all branches of life. These viruses are champions of persistence, and are maintained as proviral DNAs integrated into the genome of somatic cells and can even enter into the germ line. Infection can result in cell death, or in oncogenic transformation by insertional mutagenesis. Thus, there is tremendous evolutionary selective pressure to block or prevent retrovirus replication. In recent years, it has become apparent that mammalian cells have evolved a number of powerful mechanisms to limit or restrict virus replication, constituting novel aspects of intrinsic immunity. These mechanisms act at many diverse steps in the life cycle. The potential importance of these restriction factors is highlighted by the fact that many retroviruses, in turn, have evolved mechanisms to inactivate or overcome the blocks to infection (D. Wolf and S. P. Goff, 2008, Ann. Rev. Gen., 42: 143-163).
Integration into the genome of the host cell is a defining feature of retroviral replication. Once integrated, the viral DNA is replicated along with cellular DNA during each cycle of cell division. The viral DNA is synthesized by reverse transcription of the viral RNA genome that enters the host cell upon infection. Reverse transcription takes place in the reverse transcription complex (RTC), a nucleoprotein complex that is derived from the core of the infecting virion. The newly synthesized viral DNA remains associated with viral and cellular proteins in a large nucleoprotein complex called the pre-integration complex (PIC). The viral integrase protein that integrates viral DNA into the host genome is one of the key components of the PIC. Reverse transcription occurs in the cytoplasm, so the PIC must be transported to the nuclear periphery and cross the nuclear envelope before integration of the viral DNA into the host chromosome. The size of the PIC, which is larger than a ribosome and much larger than a nuclear pore, excludes passive diffusion as a viable mechanism for translocation, and therefore active cellular transport mechanisms must be used for this task (Y. Suzuki and R. Craigie, 2007, Nat Rev Microbiol, 5:187-196).
The synthesis of full-length viral DNA in the RTC produces the PIC. The PIC efficiently integrates viral DNA into a target DNA in vitro and is the most extensively studied retroviral nucleoprotein complex. Both MoMLV and HIV-1 PICs are large complexes with a size comparable to (or greater than) ribosomes. Information on the protein composition of PICs is mostly restricted to immunoprecipitation studies and sensitive western-blot analyses because of the small quantity of material present in cell extracts. PICs retain many components of the RTC, but differences have been reported between HIV-1 and MoMLV. Some of these differences might reflect different PIC isolation protocols and the sensitivity of assays. Immunoprecipitation of MoMLV PICs detected CA and IN22, 27, whereas HIV-1 PICs were shown to contain nucleocapsid (NC), MA, RT, IN and Vpr. In addition to viral proteins, several cellular proteins, including barrier-to-autointegration factor (BAF), high-mobility group proteins (HMGs), Ku, lamina-associated polypeptide 2 (LAP2) and lens-epithelium-derived growth factor (LEDGF/p75), have been found associated with retroviral PICs.
The chromosomal DNA that serves as the target for retroviral DNA integration is enclosed by the nuclear envelope, which forms the boundary between the nucleus and cytoplasm, and separates nucleoplasmic and cytoplasmic enzymic activities. The nuclear envelope comprises two lipid bilayers: the outer and inner nuclear membranes. These membranes are separated by a lumen and joined at nuclear pore complexes (NPCs) that serve as a gate for traffic crossing the nuclear envelope. In dividing cells, breaking and re-forming of the nuclear envelope during each cycle of cell division allows a straightforward exchange of material between the nuclear and cytoplasmic compartments. Some retroviral PICs seem to take advantage of nuclear-envelope disassembly during cell division to gain access to chromatin. However, human immunodeficiency virus 1 (HIV-1) and other lentiviruses infect non-dividing cells and must therefore cross an intact nuclear envelope. The HIV-1 PIC must therefore carry karyophilic signals that direct transport across the intact nuclear envelope through NPCs.
The ability to infect non-dividing cells is not restricted to lentiviruses. However, recent data reveal that avian sarcoma virus (ASV) as well as Friend murine leukaemia virus (FrMLV) can infect non-dividing cells including growth-arrested cells, neurons and macrophages, albeit less efficiently than HIV-1. Cell-cycle-independent infection has been also proposed for other retroviruses, including HFV and spleen necrosis virus.
In the case of HIV-1, soon after HIV-1 enters a susceptible target cell, the viral genomic RNA is reverse transcribed within the reverse transcription complex (RTC) to double-stranded DNA (S. P. Goff, Nat Rev Microbiol 5, 253 (2007)). The RTC matures into the preintegration complex (PIC), which delivers the viral DNA to the nucleus for integration into a host chromosome (Y. Suzuki, R. Craigie, Nat Rev Microbiol 5, 187 (2007)). The PIC may also sequester and protect the viral DNA from cellular DNA-modifying enzymes (K. Yoder et al., Proc Natl Acad Sci USA 103, 4622 (2006)) and from cytoplasmic DNA sensors (R. Medzhitov, Nature 449, 819 (2007); A. Takaoka et al., Nature 448:501-505 (2007); D.B. Stestson Cell 134:587-598 (2008)) that could trigger antiviral innate immunity.
Relatively little is known about the host proteins that associate with the PIC and assist in retroviral, e.g., HIV-1, integration. Retroviral integration can be divided into three steps: (1) 3′ processing (integrase (IN)-mediated hydrolysis of GT dinucleotides from HIV-1 DNA to produce reactive, recessed CAOH−3′ ends); (2) DNA strand transfer (IN-mediated insertion of the cleaved 3′ ends into opposing strands of host chromosomal DNA); and (3) 5′-end joining (repair by host enzymes of the gaps between the 5′-ends of viral DNA and the chromosome) (A. Engelman, Curr Top Microbiol Immunol 281, 209 (2003)).
While the 3′ processing of the viral DNA is required for host integration, 3′-processing also makes the viral DNA vulnerable to autointegration (C. Shoemaker et al., J Virol 40, 164 (1981); L. Li et al., J Virol 72, 2125 (1998)) in which the reactive CA ends attack sites within the viral DNA. Autointegration is mechanistically analogous to chromosomal integration, but results in nonproductive deletion or inversion circles of the viral DNA (Y. Li et al., J Virol 65, 3973 (1991); D. J. Garfinkel et al., J Virol 80, 11920 (2006); L. Li et al., J Virol 72, 2125 (1998); M. S. Lee and R. Craigie, Proc Natl Acad Sci USA 95, 1528 (1998)). Autointegration is a problem faced not only by retroviruses, but also by mobile genetic elements including bacteriophages and retrotransposons (D. J. Garfinkel et al., J Virol 80, 11920 (2006); H. W. Benjamin and N. Kleckner, Cell 59, 373 (1989); A. Maxwell et al., Proc Natl Acad Sci USA 84, 699 (1987)). Each such element employs a unique mechanism, relying on either self or host factors, to control autointegration. For example, bacteriophage Mu B protein activates DNA strand transfer to favor intermolecular transposition (A. Maxwell et al., Proc Natl Acad Sci USA 84, 699 (1987); K. Adzuma and K. Mizuuchi, Cell 57, 41 (1989)). In the case of Tn10, a cellular global regulator, H-NS, acts directly on the PIC to promote intermolecular transposition (S. J. Wardle et al., Genes Dev 19, 2224 (2005)). The barrier-to-autointegration factor (BAF) is a cellular protein that protects Moloney murine leukemia virus (MLV) PICs from autointegration and stimulates intermolecular integration in vitro (M. S. Lee and R. Craigie, Proc Natl Acad Sci USA 95, 1528 (1998); Y. Suzuki and R. Craigie, J Virol 76, 12376 (2002)). Although BAF can also stimulate HIV-1 PIC intermolecular integration activity in vitro, it has not been shown to block HIV-1 autointegration (M. C. Shun et al., J Virol 81, 166 (2007); J. M. Jacque and M. Stevenson, Nature 441, 641 (2006); H. Chen and A. Engelman, Proc Natl Acad Sci USA 95, 15270 (1998)).
Herein, we show that the SET complex plays an important role in the early phase of the retroviral lifecycle by inhibiting autointegration. Methods and compositions for inhibiting retroviral integration, and thus retroviral replication and spread of infections are described herein that exploit the identification of the role of the SET complex in preventing retroviral infection.